Coomassie brilliant blue cbb

After 18 and 25 days the osteoclasts and FBGCs were fixed for 10 min with 4% PBS buffered formaldehyde. The cells were stained for TRAcP activity (acid phosphatase leukocyte kit; Sigma-Aldrich) and with 4',6-diamidino-2-fenylindool (DAPI). The number of multinucleated cells on the bone slices were counted and related to the surface area of the bone using ImageJ. The multinucleated cells were categorized into one of the following three groups: 3 to 5 nuclei per cell, 6 to 10 nuclei per cell and more than 10 nuclei per cell.
To visualize bone resorption, bone slices were stained with coomassie brilliant blue (CBB; GE Healthcare life sciences, NJ). The bone slices were washed with milliQ water and sonicated for 30 min in 10% NH₃OH on ice. After sonification the bone slices were washed with milliQ water and transferred to a new well. The bone slices were washed in water saturated alum and incubated with water saturated alum for 10 min. After incubation the bone slices were washed twice with milliQ. Both sides of the bone chip were dried between filter paper and rinsed with CBB (10 ml) and subsequently dried between filter paper. The blue resorption pits were analyzed using ImageJ.

SDS‐PAGE analysis was conducted on 0.5 μL serum samples dissolved in PAGE sample buffer (50 mM tris‐HCL, pH 6.8 containing 50 mM dithiothreitol, 0.5% SDS, 10% glycerol) according to the equipment manufacturer's protocol (DRC Co., Ltd., Tokyo, Japan). The gel was stained with silver nitrate (Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan) or Coomassie brilliant blue (CBB) (GE Healthcare, Little Chalfont, UK), then the gel image was converted to a densitogram using Scion Image.